Project Summary Intermittently injected parathyroid hormone (PTH) is a key anabolic therapy for osteoporosis, stimulating bone formation more than resorption. Paradoxically, continuously elevated PTH causes bone loss. We have identified a factor that may explain the paradox. PTH is a potent inducer in osteoblastic lineage cells of both cyclooxygenase 2 (Cox2), the major enzyme producing prostaglandin E2 (PGE2), and receptor activator of nuclear factor ?B ligand (RANKL), which is required to commit bone marrow macrophages (BMMs) to become osteoclasts. RANKL and Cox2/PGE2 together cause BMMs to secrete a factor that suppresses the ability of continuous PTH to stimulate differentiation of osteoblasts. Using in vitro cell models, we identified the inhibitory factor as serum amyloid A3 (Saa3), an acute phase protein generally associated with inflammation or infection, and showed that Saa3 inhibited PTH-stimulated osteoblast differentiation by blocking PTH-stimulated cAMP production. As predicted by the in vitro models, PTH infusion in wild type (WT) mice suppressed bone formation and caused bone loss, but PTH infusion in in Cox2 knockout (KO) mice was markedly anabolic for bone. In contrast to the effects on formation, PTH infusion stimulated bone resorption equally in WT and Cox2 KO mice. We have generated a mouse with global KO of Saa3. We will test the hypothesis that Saa3 suppresses the anabolic, but not the catabolic, responses to continuously elevated PTH in vivo. Specific aim 1 will examine effects on bone formation and resorption of Saa3 in in vivo models for elevating PTH. Aim 2 will examine the effect of Saa3 on PTH-stimulated osteoblast and osteoclast differentiation in vitro and the cAMP- dependent actions by which PTH regulates Wnt signaling and the cAMP?independent signaling pathways by which PTH regulates RANKL signaling in our models. Aim 3 will examine the requirement for PGE2 and its EP4 receptor in the induction of Saa3 in BMMs and the role of Saa3 in stimulating other cytokines that can regulate the bone environment. We are proposing a novel role for Saa3; a new means by which osteoclasts regulate osteoblasts; and a novel role for Cox2/PGE2. The results of this study should strengthen our understanding of the molecular mechanisms underlying actions of PTH and may help us target bone remodeling more effectively to treat osteoporosis and other skeletal diseases.